Br. J. clin. Pharmac. (1979), 7,499-503

THE ACTIVITY OF ARYL HYDROCARBON HYDROXYLASE IN ADULT HUMAN SKIN P.H. CHAPMAN, M.D. RAWLINS & S. SHUSTER Departments of Pharmacological Sciences, (Clinical Pharmacology) and Dermatology, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU

1 Aryl hydrocarbon hydroxylase (AHH), a mixed-function oxidase system, has been identified in microsomal preparations of adult breast skin and foreskin. 2 Separation of dermis from epidermis by stretching, showed that AHH activity in human skin is almost exclusively located within the epidermis. 3 Preincubation of whole chopped skin with benzanthracene (5 gM to 100 giM) using a tissue culture system was accompanied by a concentration dependent increase in AHH activity. 4 Although there was no significant difference in AHH activity between the two sites, individuals differed markedly from one another in activity at any one site. Activity was observed to be positively correlated with age.

Introduction

Aryl hydrocarbon hydroxylase (AHH) (EC 1.14.14.2), a mixed-function oxidase system, has been identified in several human tissues including liver, lungs, placenta and circulating lymphocytes and monocytes (Pelkonen, 1976). AHH activity has also been described in homogenates of human neonatal foreskin (Alvares, Kappas, Levin & Conney, 1973). Since this enzyme could play an important role in determining the activity and toxicity of drugs within the skin, we have examined some of its properties in adult skin. Methods

Skin was obtained from surgical specimens of patients undergoing mastectomy (30 patients) or circumcision (18 patients). One mastectomized patient had been receiving primidone for several years prior to surgery and 13 were cigarette smokers. The patients' ages ranged from 7-83 years (mean + s.e. mean 44 + 3 years). The skin was dissected from underlying tissues and placed in ice-cold isolation medium (20 mM tris(hydroxymethyl)-methylamine in 0.3M mannitol at pH 7.4) at operation. After transfer to the laboratory, the subcutaneous fat was removed by scraping and the skin finely chopped with scissors. Chopped skin was homogenized in ice-cold isolation medium (15 ml) using a I.L.A. homogenizer. The homogenate was centrifuged at 10,000 g for 10 min and the supernatant removed and centrifuged at 100,000 g for 60 min. The microsomal pellet was resuspended in 50 mM Tris 0306-525 1/79/050499-05$01.00

chloride buffer pH 7.4 containing 3 mm MgCl2 (2 ml) and AHH activity measured in aliquots (0.5 ml) using the method of Nebert & Gelboin (1968) with benz(a)pyrene as substrate. Activity was expressed as pmol 3-hydroxybenz(a)pyrene (a generous gift from Dr H.V. Gelboin). The protein content of the resuspended microsomes was measured using the method of Lowry, Rosebrough, Farr & Randall (1951) and aqueous protein standards containing identical aliquots of Tris chloride buffer without dialysis. In some experiments, the chopped skin was preincubated for 18 h in a tissue culture system (Nebert & Gelboin, 1968) at 370C using a shaking water bath and an atmosphere of 95% 02 and 5% CO2. Varying quantities of benzanthracene were added to the culture medium before incubation, to give final concentrations ranging from 0 to 100 gM. Following the incubation, the samples were centrifuged at 10,000 g and transterred to isolation medium (15 ml) betore homogenization and assay for AHH activity as described above. Preliminary experiments showed that when carried out in this manner, no fluorescent metabolites attributable to benzanthracene interfered with the AHH assay. In four experiments, separation of dermis and epidermis was achieved by stretching. The opposite edges of specimens measuring approximately 6 cm x 4 cm were clamped, and then stretched to a tension of around 4 kg using a turn-screw attached to one of the clamps. The epidermis was removed by scraping with a scalpel, and fragments of epidermis and underlying dermis were homogenized as described above. a Macmillan Journals Ltd 1979

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0.8). No difference in basal AHH activity was observed between patients who were smokers and those who were not (t= -0.10; P> 0.9). A positive correlation, however, was seen between age and basal AHH activity in breast skin (r= +0.458; P < 0.01). (Figure 6.) Seven breast skin specimens and six foreskins were incubated in vitro with 100-200 ILM benzanthracene for 18 h and in all instances AHH activity was increased. The 'induction ratio' ('induced' activity/'basal' activity) was 3.3 + 1.1 for breast skin and 3.5 ± 1.0 for foreskin (t=0.155; P> 0.5).

AHH activity in skin Discussion Basal AHH activity was detected in homogenates of skin in all cases. Activity was found, however, to vary widely between individuals. Thus, for breast skin activity ranged from 0.90 to 6.59 pmol 3-OH-BP mg-' protein h-' (mean ± s.e. mean 3.27 + 0.21 pmol 3-OH-BP mg-' protein h-'). Foreskin AHH activity

The present study confirms the existence of AHH activity in adult human skin as well as in neonatal foreskin (Alvares et al., 1973). The results of the experiments in which AHH activity was examined in separated dermis and epidermis, suggests that the

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